Glioblastoma multiforme (GBM) is the most invasive and lethal form of brain cancer. Despite aggressive multimodal therapy, the treatment of GBM remains largely ineffective. The fundamental problem of these fatal cancers is their highly infiltrative nature and resistant property that leads to rapid tumor recurrence. The development of improved therapies targeting the infiltrating GBM cells requires new insight into the cellular and molecular mechanisms underlying GBM invasion and dispersal. We have demonstrated that a sub-population of GBM cancer cells sharing similar properties with neural stem cells displays enhanced invasive capability, angiogenic potential and therapeutic resistance, suggesting that targeting these GBM stem cells may significantly improve the treatment. Our long-term goal is to develop novel strategies targeting GBM stem cells to inhibit GBM invasion, reduce therapeutic resistance and prevent tumor recurrence. In the search for potential targets specific for GBM stem cells, we identified L1CAM as a differentially elevated surface protein in GBM stem cells relative to non-stem tumor cells and neural progenitors. Targeting L1CAM by shRNA potently disrupts neurosphere formation, suppresses tumor growth and increases the survival of mice bearing intracranial GBM xenografts. In preliminary study, we found that L1CAM knockdown in vitro remarkably reduces invasive potential of GBM stem cells, and decreases expression of three invasion-associated proteins (RhoC, Cathepsin L2 and S100A4/Metastasin), suggesting that L1CAM plays a crucial role in mediating GBM stem cell invasion, and L1CAM may represent a critical target for developing a novel anti-invasion strategy. Based on our preliminary studies, we hypothesize that L1CAM promotes GBM stem cell invasion, and molecular targeting of L1CAM in GBM stem cells in vivo inhibits tumor invasion. We will test our hypothesis and accomplish the objectives by pursuing the following three specific aims: 1. Determine the role of L1CAM in GBM stem cell invasion in vivo through inducible knockdown in xenograft models; 2. Develop therapeutic targeting of GBM stem cells using L1CAM blocking peptides (L1HM06 and L1HT08) to suppress GBM invasion and growth; 3. Define the mechanisms by which L1CAM contributes to GBM stem cell invasion using downstream overexpression rescue experiments. We will pursue these specific aims using varied molecular and cellular approaches as well as in vivo techniques such as bioluminescent imaging technology. These studies will allow us to evaluate the potential of L1CAM as a therapeutic target against GBM stem cell invasion. We expect that the outcome from these proposed studies will lay a solid foundation for future development of new regimens for treatment of GBM and other brain tumors.